1. Field of the Invention
The present invention relates to a method of stabilizing a leuco dye in a solution, a method of reducing nonspecific color development at the time of a color development reaction, and an analytical reagent composition using the methods.
2. Description of the Related Art
In the clinical examination field, various measurement methods using an enzymatic method are established. Among the measurement methods, when using an oxidase, there has been conventionally known a method which involves causing a dye to oxidatively generate in the presence of peroxidase using a generated hydrogen peroxide and various oxidation color development agents, and measuring the absorbance. For example, the combination of a coupler typified by 4-aminoantipyrine and various trinder reagents is frequently used. In contrast, as a method of detecting a smaller amount of hydrogen peroxide at high sensitivity, there has also been known a method which uses a leuco compound such as 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (ABTS). In general, such high sensitivity color development dyes, which are generally referred to as a leuco dye, have poor stability in a solution state and develop self coloring. Therefore, the duration of use is short after the preparation of a reagent, and, moreover, a reagent blank is likely to increase, which causes a problem with the measurement accuracy. Thus, such high sensitivity color development dyes have hardly been put into practical use as a clinical examination reagent kit and the like. As a method of stabilizing a leuco dye which has been reported until now, Japanese Patent Application Laid-open No. 01-118768 (Patent Document 1) reports a stabilization effect of 10-(carboxymethylaminocarbonyl)-3,7-bis(dimethylamino)phenothiazine sodium (DA-67) by cyclodextrin and the derivatives thereof. Moreover, International Patent WO 2003/033601 (Patent Document 2) discloses a method which involves causing peroxidase or fructosyl amino acid oxidase to coexist so as to stabilize a leuco dye N-(carboxymethylaminocarbonyl)-4,4′-bis(dimethylamino)biphenylamine sodium (DA-64). Further, Japanese Patent Application Laid-open No. 2005-110507 (Patent Document 3) discloses a method of stabilizing N,N,N′,N′,N″,N″-hexa(3-sulfopropyl)-4,4′,4″-triaminotriphenylmethane-6Na (TPM-PS).
According to Japanese Patent Application Laid-open No. 01-118768 (Patent Document 1), although the stabilization effect when stored for a short period of time (ten days or shorter) is recognized, there is no disclosure about the behavior when stored for a long period of time. Thus, the stabilization effect when stored for a long period of time is problematic. Further, according to International Patent WO 2003/033601 (Patent Document 2), although it is determined that a dye is stable only based on a spectrum of wavelength, there is no disclosure about whether the quantity of hydrogen peroxide has been actually determined successfully using this. Further, according to Japanese Patent Application Laid-open No. 2005-110507 (Patent Document 3), the stabilization effect by a specific buffer solution and a specific chelating agent is shown. However, the absorbance after stored for a short period of time is measured, and then the stabilization effect after one year is estimated simply by performing proportional calculation using the absorbance. Therefore, the basis for determining the stabilization effect is not sufficient, and the stabilizing effect when actually stored for a long period of time is in doubt.
In contrast, even when stabilization is achieved by a single use of a leuco dye, there is a problem that the dye develops color in a nonspecific manner at the time of the color development reaction of hydrogen peroxide to which peroxidase has been added, which impairs exact measurement. More specifically, in a state where a leuco dye and peroxidase coexist at the time of the color development reaction of hydrogen peroxide, there is a problem that a reagent blank increases with time by a nonspecific reaction even when hydrogen peroxide does not exist.
Since the leuco dye is suitable for high sensitivity detection of hydrogen peroxide, a method of stabilizing the leuco dye which is stable in a liquid state for a long period of time is desired.
[Patent Document 1] Japanese Patent Application Laid-open No. 01-118768
[Patent Document 2] WO 2003/033601
[patent Document 3] Japanese Patent Application Laid-open No. 2005-110507